A Chemo-Enzymatic Approach Towards Gamma-Thio-Nucleoside-5'-Triphosphates

Context of Proposed Research
Our methods will give a simpler route towards nucleoside-5'-triphosphates (NTPs), which are key molecules in a range of biotechnological and basic research applications. Currently there are two main approaches. The first is readily accessible to biologists, but offers only small amount of material at high cost. The other requires highly trained chemists to provide the materials, where the chemistry is challenging and often unreliable. Our approach represents a new biotechnology

Aims and Objectives
We aim to provide two related simpler approaches that should allows biologists to access the desired NTPs with relative easy in also allow them to access more useful amounts of these materials in a cost effective manner. Our objectives centre on using an enzyme system, nucleoside diphosphate kinase (NDPK), to facilitate the production of NTPs.

Potential Applications and Benefits
The specific class of NTP that we will address in this one-year proof-of-concept proposal has a range of applications in determining how enzymes work and also how signaling pathway operate within biological systems. Our approach will benefit research workers in these areas and the specialist chemical companies that prepare NTPs. With our concept proven, we hope to expand our strategy towards a broader range of nucleotide systems, and generate a generic, broad platform towards this class of molecule.

The five technical objectives will be addressed as follows:

OBJECTIVE 1: ACCESS ACTIVE NUCLEOSIDE DIPHOPHATE KINASES (NDPKs)
His-tagged WT NDPKs and His->Gly active site mutants will be expressed, and activities will be assessed using HPLC, where the rate of equilibration between UTP and ATP will be measured (imidazole will be added to rescue His->Gly mutants).

OBJECTIVE 2: EXPLORE IMIDAZOLE THIOPHOSPHORYLATION AND Im RESCUE OF -HIS MUTANT NDPKs
Literature shows that His->Gly NDPK competently transfers the gamma-thio-P group of gamma-thio-ATP to imidazole: we envisage the reverse process will proceed smoothly. Thiophosphorylated imidazole will be prepared, exposed to NDPK plus ADP, and gamma-thio-ATP generation will be confirmed by HPLC. Thereafter, imidazole will be phosphorylated in situ and the thiophosphorylation of ADP will be measured.

OBJECTIVE 3: EXPLORE DIRECT HIS THIOPHOSPHORYLATION OF WT NDPKs
Immobilsed NDPK will be exposed to thiophosphodichloridate ion or thiophosphoryl chloride to generate thiophosphorylated-NDPK. Phosphoryl transfer activity of this NDPK to ADP will be measured by HPLC, and protein thiophosphorylation will be confirmed by MS. These procedures will be repeated concurrently to demonstrate a continuous process.

OBJECTIVE 4: EXPLOIT THE MOST PROMISING RESULTS FROM OBJECTIVE 2 & 3 ACROSS A RANGE OF COMMERCIAL NDP SUBSTRATES.
HPLC will be used to screen a range of NDP substrates, and products will be compared to reference compounds and/or confirmed using MS and 31-P NMR methods

OBJECTIVE 5: CONFIRM UTILITY OF THIO-NTP SYSTEMS
NTP-based enzyme systems avilable in each of our labs will be assayed to gauge the 'quality' of representative gamma-thio-NTPs. These will include T7 RNA polymerase (DRWH lab), adenylyl cyclases (MJC lab), and Ire1 systems (MS lab). NDPK will also serve as its own test vehicle, where materials generated by NDPK will be re-assayed in the reverse direction via HPLC.

Academic and industrial researchers will benefit from this research. Our approach will make important research and biotechnology chemicals more readily available to a wider range of workers. We shall protect IP and engage with industrial users in order to allow reap economic benefits. Our research will be published to allow the general research community to benefit from our findings.